ROMK channel was the first of what is now a large family of two- transmembrane containing, inwardly rectifying K+ channels that have been cloned. It encodes the low conductance K channel identified in the apical membranes of distal nephron segments. Recent functional studies have shown that ROMK channel activity is regulated by phosphorylation- dephosphorylation processes, and is sensitive to nucleotide binding as well as the ATP/ADP ratio, a defining character for KATP channels. In addition, mutations at the putative nucleotide-binding motif alter the channel's sensitivity to nucleotide. I propose to perform biochemical analysis to (l).Establish that ROMK peptides bind nucleotides, and examine the ability of other nucleotides (ADP, AMP-PNP, AMP, UTP, GTP, etc.) to compete for binding. (2). Assess the effects of salt concentration, pH and PKA-dependent phosphorylation on nucleotide binding. (3). Determine amino acid residues that directly interact with nucleotides.